Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, and limit concentration 100 mg/L (nominal) - Sampling method: For the determination of the actual test item concentration, duplicate samples were taken at the start and end of each test medium renewal periods from the test concentration and from the control. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling. Immediately after sampling, methanol (10 mL methanol per 100 mL sample volume) was added to each sample to stabilize the latter during the storage period. The concentration of the test item 10-HSA was analytically measured in the duplicate test medium samples of the single test concentration taken at the start and end of each test medium renewal period. From the control, one of the duplicate samples was analyzed per sampling time. - Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION- Method: Due to the low water solubility of the test item, a suspension with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 100 mg of the test item (dosing range: 100.02 and 100.05 mg) in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer for three hours at room temperature in the dark to dissolve a maximum amount of the test item in the test water. After stirring, the suspension of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The undiluted filtrate with a loading rate of 100 mg/L was used as test medium. The test medium was prepared just before the introduction of the daphnids (i.e., start of the test) and prior to the test medium renewal after 24 hours.- Controls: yes, test water without test item. - Evidence of undissolved material: The test medium was a clar solution throughout the entire test duration.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM - Common name: water flea - Strain/clone: originally from the Daphnia Collection of the University of Basel, Switzerland (2015) - Source: in-house culture at IES Ltd Laboratories - Age of daphnia at the start of the test: 6 - 24 h old and not first brood progeny - Feeding during test: no

BREEDING: - Food type: algal suspension of the green algae Desmodesmus subspicatus, strain n° 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen, Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal supension and a commercial fish diet (Tetra Min Hauptfutter, TETRA-Werke, Melle, Germany) - Frequency: three times a week

ACCLIMATION - No, the daphnia were bred under temperature and light conditions identical to those of the tests.

TEST SYSTEM - Test vessel: beakers - Type: The beakers were covered with transparent, disposable polypropylene sheets to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions. - Material, size, fill volume: Material: glass; Size: 100 mL; Fill volume: 50 mL - Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. - Renewal rate of test solution: after 24 h (one time during entire test) - No. of organisms per vessel: 5 - No. of vessels per concentration (replicates): 4 - No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS - Source/preparation of dilution water: Reconstituted test water, consisting of analytical grade salts dissolved in purified water according to ISO 6341 - Alkalinity: 0.8 mmol/L - Ca/mg ratio: 4:1 (based on molarity) - Na:K ratio: 10:1 (based on molarity) - Culture medium different from test medium: no - Intervals of water quality measurement: At the start and end of each test medium renewal period, the pH values, dissolved oxygen concentration and water temperature were determined in each treatment. In addition, the appearance of the test medium was visually controlled and documented.

RANGE-FINDING STUDY - Test concentrations: A stirring pre-experiment (GLP) with three individual suspensions of 100 mg/L test item (nominal) with different stirring regimes (3, 24, and 96 h) was performed to determine the solubility of the poorly soluble test item (water solubility < 100 mg/L) in test water and to determine the optimum time period to reach equilibration within a reasonable time period. - Results used to determine the conditions for the definitive study: The analytically determined concentration of dissolved test item in the filtrates were 0.36 mg/L (3 h), 0.25 mg/L (24 h), and 0.26 mg/L (96 h). These results showed that the maximum concentration of dissolved test item in test water was reached after a stirring period of 3 h.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0% - Effect concentrations exceeding solubility of substance in test medium: yes, the analytically measured concentration of 0.26 mg/L corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L (nominal).

The 24-hour and 48-hour EC/EL-values could not be calculated due to the absence of a toxic effect of the test item. Therefore, the EC0/EL0, EC50/EL50, EC100/EL100 and the NOEC/NOEL were directly determined from the raw data.

Any other information on results incl. tables

RESULTS OF EXPERIMENTAL CONDITIONS

Table 1. pH Values in the Treatments.

Mean measuredtest itemconcentration

Exposure time

0 h

24 h

48 h

[mg/L]

new

old

new

old

Control

7.8

7.9

7.9

7.8

0.26

7.6

7.8

7.7

7.8

Table 2. Dissolved Oxygen Concentrations [mg/L] in the Treatments.

Mean measuredtest itemconcentration

Exposure time

0 h

24 h

48 h

[mg/L]

new

old

new

old

Control

8.7

8.5

8.6

8.4

0.26

8.1

8.3

8.2

8.5

ANALYTICAL RESULTS

The test item concentration remained stable during the test (Table 3).

Table 3. Results for Test Samples

Sampling Day /Sample Age

Loading Rate of 100 mg Test Item/L

Measured Concentrationof Test Item x

Sample Preparation FactorF

Determined Concentration of Test Itemc

Sample 1

Sample 2

Mean Value

[day/hours]

[mg/L]

[mg/L]

[mg/L]

[mg/L]

0/0

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(fresh)

Undiluted Filtrate

2.62

2.47

2.54

0.100

0.254

1/24

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(aged)

Undiluted filtrate

2.41

2.89

2.65

0.100

0.265

1/0

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(fresh)

Undiluted Filtrate

2.60

2.46

2.53

0.100

0.253

2/24

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(aged)

Undiluted Filtrate

2.72

2.48

2.60

0.100

0.260

LOQ= 0.0145 mg test item /L

n.d.= not detected

n.a.= not applicable

No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solutions throughout the entire test duration.

The biological results were related to the mean measured test item concentration of 0.26 mg/L and to the loading rate of 100 mg/L.

BIOLOGICAL RESULTS

During the test period of 48 h, no immobilized test organisms were determined in the control and the limt test concentration of 0.26 mg/L (loading rate of 100 mg/L).

Table 4. Effect of test item on the Mobility of Daphnia magna.

Mean measuredtest itemconcentration

No. ofdaphnidstested

Immobilizeddaphnids after24 hours

Immobilizeddaphnids after24 hours

Immobilizeddaphnids after48 hours

Immobilizeddaphnids after48 hours

[mg/L]

No.

[%]

No.

[%]

Control

20

0

0

0

0

0.26*

20

0

0

0

0

*:Undiluted filtrate of a suspension with a loading rate of 100 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item 10-HSA had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to the analytically measured concentration of 0.26 mg/L which corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L.

Executive summary:

The acute toxicity of the test item10-HSA to Daphnia magna was determined in a 48‑hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

Due to the low water solubility of the test item, a suspension of the test item with the loading rate of 100 mg/L was prepared by using ultrasonic treatment for 15 minutes and intensive stirring for 3 hours to reach a maximum concentration of dissolved test item in test water. The stirring time was based on a pre-experiment. After stirring, the suspension was filtered through a 0.45 µm membrane filter. The undiluted filtrate was used as test medium.

The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including the loading rate of 100 mg/L. Thus, the only loading rate tested was 100 mg/L. Additionally a control was tested in parallel.

The measured concentration of 10-HSA in the test medium with a loading rate of 100 mg/L was 0.25 mg/L at the start of both renewal periods. At the end of the two 24-hour renewal periods, 103 and 104 % of the initially measured concentration were found. Thus, the test item was sufficiently stable during the test medium renewal periods of 24 hours. The mean measured concentration during the test period of 48 hours was calculated to be 0.26 mg/L (arithmetic mean over all measurements of test concentration).

The biological results were related to the mean measured test item concentration of 0.26 mg/L and to the loading rate of 100 mg/L:

24-hour EC50/EL50 > 0.26/ >100 mg/L

24-hour EC0/EL0 ≥0.26 /≥100 mg/L

24-hour EC100/EL100 > 0.26 / > 100 mg/L

48-hour EC50/EL50 > 0.26 / > 100 mg/L

48-hour EC0/EL0 and 48-hour NOEC/NOEL ≥0.26 / ≥100 mg/L

48-hour EC100/EL100 > 0.26 / > 100 mg/L

In conclusion, the test item 10-HSA had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to the analytically measured concentration of 0.26 mg/L which corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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